Discovery of Small Molecule Bak Activator for Lung Cancer Therapy.

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

In Vitro and In Vivo Synergistic Antitumor Activity of the Combination of BKM120 and Erlotinib in Head and Neck Cancer: Mechanism of Apoptosis and Resistance.

We previously reported that the EGFR-targeted inhibitor erlotinib induces G1 arrest of squamous cell carcinoma of the head and neck (SCCHN) cell lines without inducing significant apoptosis. Large-scale genomic studies suggest that >50% of SCCHN cases have activation of PI3K pathways. This study investigated whether cotargeting of EGFR and PI3K has synergistic antitumor effects and apoptosis induction. We examined growth suppression, apoptosis, and signaling pathway modulation resulting from single and combined targeting of EGFR and PI3K with erlotinib and BKM120, respectively, in a panel of SCCHN cell lines and a xenograft model of SCCHN. In a panel of 12 cell lines, single targeting of EGFR with erlotinib or PI3K with BKM120 suppressed cellular growth without inducing significant apoptosis. Cotargeting of EGFR and PI3K synergistically inhibited SCCHN cell line and xenograft tumor growth, but induced variable apoptosis; some lines were highly sensitive, others were resistant. Mechanistic studies revealed that the combination inhibited both axes of the mTORC1 (S6 and 4EBP1) pathway in apoptosis-sensitive cell lines along with translational inhibition of Bcl-2, Bcl-xL, and Mcl-1, but failed to inhibit p-4EBP1, Bcl-2, Bcl-xL, and Mcl-1 in an apoptosis-resistant cell line. siRNA-mediated knockdown of eIF4E inhibited Bcl-2 and Mcl-1 and sensitized this cell line to apoptosis. Our results strongly suggest that cotargeting of EGFR and PI3K is synergistic and induces apoptosis of SCCHN cell lines by inhibiting both axes of the AKT-mTOR pathway and translational regulation of antiapoptotic Bcl-2 proteins. These findings may guide the development of clinical trials using this combination of agents. Mol Cancer Ther; 16(4); 729-38. ©2017 AACR.

Preclinical In Vitro, In Vivo, and Pharmacokinetic Evaluations of FLLL12 for the Prevention and Treatment of Head and Neck Cancers.

Despite its high promise for cancer prevention and therapy, the potential utility of curcumin in cancer is compromised by its low bioavailability and weak potency. The purpose of the current study was to assess the in vitro and in vivo efficacy and pharmacokinetic parameters of the potent curcumin analogue FLLL12 in SCCHN and identify the mechanisms of its antitumor effect. IC50 values against a panel of one premalignant and eight malignant head and neck cancer cell lines as well as apoptosis assay results suggested that FLLL12 is 10- to 24-fold more potent than natural curcumin depending on the cell line and induces mitochondria-mediated apoptosis. In vivo efficacy (xenograft) and pharmacokinetic studies also suggested that FLLL12 is significantly more potent and has more favorable pharmacokinetic properties than curcumin. FLLL12 strongly inhibited the expression of p-EGFR, EGFR, p-AKT, AKT, Bcl-2, and Bid and increased the expression of Bim. Overexpression of constitutively active AKT or Bcl-2 or ablation of Bim or Bid significantly inhibited FLLL12-induced apoptosis. Further mechanistic studies revealed that FLLL12 regulated EGFR and AKT at transcriptional levels, whereas Bcl-2 was regulated at the translational level. Finally, FLLL12 strongly inhibited the AKT downstream targets mTOR and FOXO1a and 3a. Taken together, our results strongly suggest that FLLL12 is a potent curcumin analogue with more favorable pharmacokinetic properties that induces apoptosis of head and neck cancer cell lines by inhibition of survival proteins including EGFR, AKT, and Bcl-2 and increasing of the proapoptotic protein Bim.

Activation of muscarinic cholinoceptor ameliorates tumor necrosis factor-α-induced barrier dysfunction in intestinal epithelial cells.

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Comparison of subcellular distribution and functions between exogenous and endogenous M1 muscarinic acetylcholine receptors.

AIMS: Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors. MAIN METHODS: Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7). KEY FINDINGS: M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca(2+) mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca(2+) mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs. SIGNIFICANCE: Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. METHODS: We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets. RESULTS: The mAChRs were detected on the crypts (K d = 163.2 ± 32.3 pM, B max = 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium. CONCLUSIONS: These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.